Abstract The aim of the study was to evaluate the kinetic parameters of a specific serotonin transporter SERT and serotonin uptake in a mentally healthy subset of patients with fibromyal
Trang 1Open Access
Vol 8 No 4
Research article
Alteration of serotonin transporter density and activity in
fibromyalgia
Laura Bazzichi1, Gino Giannaccini2, Laura Betti2, Giovanni Mascia2, Laura Fabbrini2, Paola Italiani2, Francesca De Feo2, Tiziana Giuliano1, Camillo Giacomelli2, Alessandra Rossi2,
Antonio Lucacchini2 and Stefano Bombardieri1
1 Department of Internal Medicine, Division of Rheumatology, University of Pisa, Via Roma 67 - 56126 PISA Italy
2 Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy
Corresponding author: Antonio Lucacchini, lucas@farm.unipi.it
Received: 21 Feb 2006 Revisions requested: 30 Mar 2006 Revisions received: 12 May 2006 Accepted: 31 May 2006 Published: 21 Jun 2006
Arthritis Research & Therapy 2006, 8:R99 (doi:10.1186/ar1982)
This article is online at: http://arthritis-research.com/content/8/4/R99
© 2006 Bazzichi et al.; licensee BioMed Central Ltd
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The aim of the study was to evaluate the kinetic parameters of a
specific serotonin transporter (SERT) and serotonin uptake in a
mentally healthy subset of patients with fibromyalgia Platelets
were obtained from 40 patients and 38 healthy controls SERT
expression and functionality were evaluated through the
measurement of [3H]paroxetine binding and the [3H]serotonin
uptake itself The values of maximal membrane binding capacity
(Bmax) were statistically lower in the patients than in the healthy
volunteers, whereas the dissociation constant (Kd) did not show
any statistically significant variations Moreover, a decrease in
the maximal uptake rate of SERT (Vmax) was demonstrated in the
platelets of patients, whereas the Michaelis constant (Km) did not show any statistically significant variations Symptom severity score (tiredness, tender points index and Fibromyalgia
Impact Questionnaire) were negatively correlated with Bmax and
with Vmax, and positively correlated with Km A change in SERT seems to occur in fibromyalgic patients, and it seems to be related to the severity of fibromyalgic symptoms
Introduction
Fibromyalgia syndrome (FMs) is a chronic pain syndrome
char-acterized by widespread pain and stiffness, multiple tender
points, and fatigue [1] This pain syndrome has an incidence
of 2% in the general population and occurs with higher
fre-quency among women in middle age [2] FM is often
associ-ated with increased prevalence of depressive symptoms,
major depression and anxiety [3] The cause and
pathophysi-ology of FMs is unclear; pathophysiological hypotheses
include impairment in the functioning of the
hypothalamic-pitu-itary axis and alterations in specific neurotransmitters such as
substance P, N-methyl-D-aspartate, noradrenaline
(norepine-phrine) and serotonin (5-HT) However, interest has been
growing in a possible involvement of 5-HT in FM Indeed,
strong evidence has accumulated to support the hypothesis
that a deficiency in serotonergic neuronal functioning might be
related to the pathophysiology of FM [5-8] Patients with FM
have been found to have decreased concentrations of 5-HT and tryptophan (5-HT precursor) in serum and cerebrospinal fluid 5-HT, in particular, is theorized to have a function in stage
4 sleep and in the pain threshold [9] This neurotransmitter is implicated in psychiatric disorders such as depression, anxi-ety, and obsessive compulsive disorder Stressful experiences lead to depression in some people who are already genetically predisposed, and increase the probability of FM exordium The 5-HT gene could moderate the serotonergic response to stress [4]
As a mediator, 5-HT exerts its actions by means of interaction with distinct receptors, which are differentiated on the basis of structures, molecular mechanisms and pharmacological pro-files [9] On this basis, drugs acting on 5-HT receptors, in par-ticular on 5-HT2 and 5-HT3, are being used or investigated for the clinical management of FM [10,11] Among drugs
5-HT = serotonin; Bmax = maximal membrane binding capacity; FIQ = Fibromyalgia Impact Questionnaire; FM = fibromyalgia; Kd = dissociation
con-stant; Km = Michaelis constant; SERT = specific serotonin transporter; SSRI = selective 5-HT reuptake inhibitor; TPi = tender point index; Vmax = maximal uptake rate of SERT.
Trang 2targeting 5-HT receptors, ketanserin is a selective 5-HT2
antagonist that can reduce the hyperalgesia, spontaneous
pain, sleep disorders and other symptoms of FM [12], and
granisetron and tropisetron are selective 5-HT3 receptor
antagonists that show clinical efficacy in FM [11]
In an analogous manner to other transmitters, the endogenous
activity of 5-HT is controlled by a specific 5-HT transporter
(SERT), which mediates the intracellular reuptake of 5-HT and
can be specifically blocked by selective 5-HT reuptake
inhibi-tors (SSRIs) such as paroxetine and fluoxetine SERT is widely
expressed in intestinal epithelial cells, in central or peripheral
serotonergic neurons and in platelets; it shares common
molecular and physiological features in these locations
[13-15] Clinical studies have demonstrated the efficacy of SSRI
in FM [16], but the data are not unequivocal Nociception
refers to the physiological process of transmitting a painful
stimulus from the periphery through afferent neurons to the
cerebral cortex It has been postulated that serotonergic
neu-rotransmission has a significant function in nociception
[17,18]; alterations in 5-HT metabolism and transmission
might therefore be important in the pathogenesis of FM These
findings support the proposal that aberrant pain perception in
FM also results from an instability of the 5-HT system in FM
There is also evidence that changes in the expression of SERT
are due to a polymorphism in the transcription region in
patients with FM [19]
In the present study, both the expression and functionality of
SERT were determined in platelets collected from patients
with FM, with the following aims: first, to perform a comparison
with the pharmacological profile of platelet SERT in healthy
volunteers, and second, to examine putative correlations of
SERT characteristics with the severity of symptoms such as
tiredness, Fibromyalgia Impact Questionnaire (FIQ) score or
tender point index (TPi)
Materials and methods
Materials
[3H]Serotonin (specific radioactivity 30 Ci/mmol) and [3
H]par-oxetine (specific radioactivity 19.1 Ci/mmol) were purchased
from Perkin-Elmer Life Science (Milano, Italy) All other
rea-gents were obtained from normal commercial sources
Subjects
Forty patients (all female) affected by primary FM, aged 53 ±
13 years (mean ± SD) took part in the study, and 38 healthy
females age-matched to the patients (50 ± 12 years) were
used as a control group The American College of
Rheumatol-ogy criteria for FM [1] were used to make the diagnosis of FM
The inclusion criteria for the study groups comprised a
nega-tive history for psychoacnega-tive drug treatment and other
neuro-logical disorders None of the subjects had comorbid
psychiatric disorders or had received treatment with
antide-pressant drugs No patient was under pharmacological
treat-ment All patients underwent a wash-out period of 2 months before the study They were enrolled at the University Division
of Rheumatology, Santa Chiara Hospital, Pisa Written con-sent was obtained from all patients and controls after a full explanation of the procedure The study was approved by the local Ethics Committee
Evaluation of clinical parameters
For each patient and control, tenderness at tender points was evaluated by means of the Fischer dolorimeter [20] The total fibromyalgic tender point score (right plus left) was used for statistical analysis Each positive tender point had a pain score between 0 (no pain) and 3 (severe pain) We also calculated the TPi as the sum of the scores of all tender points divided by the total number of tender points
To estimate the impact of FM on the quality of life, all patients and controls received an FIQ [21] The total score was the sum of the values of the 10 FIQ items, which reflected the impact of FM and ranged from 0 (no impact) to 100 (maximum impact)
Tiredness was evaluated as measured by the FIQ tiredness item, which consisted of a visual analogic scale numbered between 0 and 10
To exclude major psychiatric disorders, all patients were eval-uated by means of a diagnostic interview consisting of the administration of the Structured Clinical Interview for DSM-IV axis-I disorder (SCID-I/P) [22] This assessment was con-ducted by psychiatrists who were trained and certified in the use of the study instruments at our department
Separation of platelets
Venous blood (30 ml) was collected from each subject and gently mixed with 1 ml of anticoagulant (0.15 M EDTA) Plate-let-rich plasma was obtained by low-speed centrifugation
(200g for 20 minutes at 22°C) Platelets were counted
auto-matically with a flux cytometer (Cell-dyn 3500 system; Abbott, Milano, Italy)
For measurement of [3H]serotonin reuptake, platelets were used immediately, whereas for [3H]paroxetine binding,
plate-lets were precipitated by centrifugation at 10,000g for 10
min-Table 1 [ 3 H]Paroxetine binding characteristics in platelet membranes
of control subjects and patients with fibromyalgia
Results are shown as means ± SEM Bmax, maximal membrane
binding capacity; FM, fibromyalgia; Kd, dissociation constant ap <
0.0001.
Trang 3utes at 4°C and the pellets were then stored at -80°C until the
assay
[ 3 H]Serotonin uptake
[3H]Serotonin uptake was performed as described by Arora
and Meltzer [23], with some modifications Aliquots of
plate-lets (2 × 106 platelets) were incubated for 10 minutes at 37°C
with [3H]serotonin at six concentrations from 15 to 700 nM in
an assay buffer (118 mM NaCl, 4.7 mM KCl, 1.07 mM
MgSO4.7H2O, 1.17 mM KH2PO4, 25 mM NaHCO3, 11.6 mM
glucose, pH 7.4, in the presence of 0.1% ascorbate and 100
µM Pargyline) to a final volume of 0.5 ml Non-specific uptake
was measured in the presence of 10 µM Fluoxetine
Preparation of platelet membranes
The platelet pellets were washed with 10 ml of buffer (150 mM
NaCl, 20 mM EDTA, 50 mM Tris-HCl, pH 7.4) Pellets were
lysed and homogenized in 10 ml of buffer (5 mM Tris-HCl, 5
mM EDTA, pH 7.4, containing the following protease
inhibi-tors: 200 µg/ml bacitracin, 160 µg/ml benzamidine and 20
µg/ml soybean trypsin inhibitor) with an Ultra-Turrax
homoge-nizer and centrifuged twice at 30,000g for 15 minutes at 4°C.
The resulting pellets were resuspended in ice-cold 50 mM
Tris-HCl buffer, pH 7.4, containing protease inhibitors, and
centrifuged at 50,000g for 15 minutes at 4°C The pellets
were then suspended with assay buffer (50 mM Tris-HCl, 120
mM NaCl, 5 mM KCl, pH 7.4)
Protein concentration was determined with the method of
Lowry and colleagues [24] using bovine serum albumin as a
standard
[ 3 H]Paroxetine binding assay to platelet membranes
[3H]Paroxetine binding was performed as described by
Marazziti and colleagues [25] The incubation mixture
con-sisted of 100 µl of platelet membranes (50 to 100 µg of
pro-tein per tube), 50 µl of [3H]paroxetine at six concentrations
(0.01, 0.025, 0.05, 0.25, 0.5 and 1 nM) and 1.85 ml of assay
buffer Specific binding was obtained by using 10 µM
fluoxet-ine as a displacer
Data analysis
Equilibrium-saturation binding data, the maximum binding
capacity (Bmax, fmol/mg of protein) and the dissociation
con-stant (Kd, nM) were analysed by means of the iterative
curve-fitting computer programs EBDA and LIGAND (Kell for
Win-dows, v 6.0)
The maximal uptake rate of SERT (Vmax, pmol/109 cells per
minute) and the Michaelis constant (Km, nM) were obtained by
direct weighted nonlinear regression of uptake rate against
[3H]serotonin concentration with GraphPad PRISM software
(GraphPad, San Diego, CA, USA)
Statistical analysis was performed with Student's t test The
relationship between variables was checked with a two-tailed Spearman analysis
Results
[3H]Paroxetine binding sites on the SERT of platelet mem-branes were used as a first marker of serotonergic function
Table 1 shows the mean Bmax values for the density, and Kd val-ues for the affinity, of [3H]paroxetine binding on platelets in the patients and in the healthy controls A statistically significant
difference was found between the two groups for Bmax (1,048
± 30 versus 1,260 ± 34 fmol/mg for control; mean ± SEM) but
not for Kd (0.086 ± 0.021 nM versus 0.077 ± 0.012 nM for control; mean ± S.E.M.)
The evaluation of symptom severity in patients with FM (Table 2) yielded tiredness scores from 2 to 10 with a mean of 7.4 ± 2.2 (SD), TPi values from 1 to 3 with a mean of 2.2 ± 0.5 (SD), and FIQ scores from 8 to 94 with a mean of 57.1 ± 21.3 (SD) The severity of symptoms was shown to be related to the max-imal binding capacity of platelet SERT, in that the statistical analysis showed a negative correlation between symptom
scores and the respective Bmax values (p < 0.0001 for tired-ness; p < 0.0001 for TPi and p < 0.0001 for FIQ) By contrast,
no significant correlation was found when comparing
symp-tom severity with Kd values
Age did not seem to influence the binding parameters of plate-let SERT in either patients with FM or healthy volunteers [3H]Serotonin uptake on platelets was used as marker of the
functionality of SERT Vmax and Km values of [3H]serotonin uptake in patients with FM accounted for 90 ± 4 pmol/109
platelets per minute and 96 ± 15 nM, respectively (means ± SEM; Table 3) A comparison of these parameters with those
in healthy volunteers indicated significant differences for Vmax values but not for Km values: whereas the mean Vmax in the patients with FM was significantly lower (90 ± 4 versus 114 ±
6 pmol/109 platelets per minute for controls; p < 0.001), the mean Km in the patients with FM was not significantly different (96 ± 15 versus 114 ± 15 nM for controls; Table 2)
The severity of the symptoms was related to Vmax and Km in that the statistical analysis showed a negative correlation between
the symptom scores and the respective Vmax values (p < 0.0001 for tiredness; p < 0.05 for TPi and p < 0.001 for FIQ).
We also found a positive correlation between Km values and
tiredness (p < 0.001), TPi (p < 0.001) and FIQ (p < 0.001).
The covariate age was shown not to have an effect on the
var-iability of the Vmax or Km values in either patients with FM or healthy volunteers
Discussion
The present study is the first examination of the relationships between SERT expression and kinetic parameters of 5-HT
Trang 4uptake We have found statistically significant differences in
Bmax and Vmax values between patients with FM and controls
Moreover, we found a negative correlation between symptom
scores and the respective Bmax and Vmax values (Bmax and Vmax
versus tiredness, TPi and FIQ score) The platelet is
consid-ered to be a peripheral model of neuronal activity with respect
to 5-HT function In fact, previous studies have demonstrated
that the same SERT is expressed in the central nervous
sys-tem and platelets [14] Moreover, the identity between the two
structures, as confirmed by sequence homologies through
cloning studies [15], has provoked a surge of different studies
in neuropsychiatric disorders, given the possibility of exploring
peripherally a mechanism of the central nervous system [26]
Recently it has been proposed that altered serotonergic
neu-ronal function might be related to the pathophysiology of FM
[5,8,27] These findings prompted us to investigate the
char-acteristics of SERT in the platelets of patients with FM Bmax
and Kd values of [3H]Paroxetine binding were assumed to
rep-resent SERT density and ligand binding affinity, respectively,
whereas Vmax and Km values of [3H]Serotonin uptake were
taken as estimates of SERT rate and affinity, respectively A
very interesting observation was that both binding and uptake
parameters differed significantly from those of healthy
volunteers
The patients with FM have fewer SERTs expressed on the
cel-lular membrane than healthy subjects (a decreased Bmax,
per-haps because the SERTs are less transcribed) Besides
having fewer SERTs, patients with FM have a deficit in
func-tionality (demonstrated by a decrease in transport rate)
Such combined changes in Bmax and Vmax values allow the
inference that the efficiency of 5-HT uptake by platelet SERT
is altered Our previous studies demonstrated an alteration of
SERT density and of the uptake rate of SERT in psychiatric
patients [28,29] Consistent with this suggestion was the
cor-relation analysis in the present study: the lower the density and
rate of SERT on platelet membranes, the higher the severity of
FM symptoms Moreover, the Km values were also positively
correlated with tiredness, TPi and FIQ
A reduced density and rate of SERT are consistent with previ-ous observations indicating that levels of 5-HT are altered in patients with FM [30,31] The biophysiological mechanism of
FM has been proposed to be similar to that in depression, and
it has been suggested that this is likely to result from a neu-roendocrine/neurotransmitter dysregulation [32] However,
we suppose that the alterations in Bmax and Vmax values are not related to the pathophysiology of FM but are a consequence
of FM Our hypothesis is that a decrease in Bmax and Vmax of SERT is due to a pain stimulus [33,34]
It has been shown that the decreased pain perception thresh-old during depression is likely to result from a dysfunction in several neurotransmitter systems, especially the serotoniner-gic one, which is also involved in the pathophysiology of depression [35] In addition, an excessive stimulation of peripheral 5-HT receptors would account for pain and might explain why the clinical use of 5-HT3 receptor antagonists such as tropisetron or granisetron can promote the relief of disturbance associated with FM [11,36]
SERT has been investigated previously in patients with FM, with discordant results Russell and colleagues [37] found a
higher Bmax in patients with FM than in healthy controls,
whereas other authors found normal Bmax values [28,35] using [3H]Paroxetine or [3H]Imipramine [38] In our experiments we used [3H]Paroxetine, which binds with high affinity to a spe-cific population of binding sites located on human platelets and neuronal membranes, associated with 5-HT uptake mech-anisms [39] The present results indicate a decrease in the density and rate of platelet SERT in patients with FM, and allow us to propose a specific role for SERT in the pathogen-esis of FM In fact, we avoided the inclusion of patients with
FM who had psychiatric components because it is known that
in psychiatric disorders such as depression, the expression of SERT is altered [40-42] and it is very difficult to identify the role of the two components in patients with FM who have
comorbid psychiatric disorders Thus, the changes in Bmax and
Vmax demonstrated in our study may be due to FM only
There is also a possible contribution from 5-HT to the aetiology
of FM because of the efficacy of SSRIs in the management of chronic pain in idiopathic pain disorders [43] Thus, in view of
the decreased Bmax and Vmax values found in our subset of
Table 2
Characteristics of control subjects and patients with
fibromyalgia
Results are means ± SD FIQ, Fibromyalgia Impact Questionnaire;
FM, fibromyalgia; TPi, tender point index.
Table 3 Serotonin uptake characteristics in platelets of control subjects and patients with fibromyalgia
a
Results are shown as means ± SEM FM, fibromyalgia; Km, Michaelis
constant; Vmax, maximal uptake rate of specific serotonin transporter
ap < 0.001.
Trang 5mentally healthy patients, who were SSRI free, we propose
that there is a compensatory mechanism in the central nervous
system to relieve the pain This may clarify the improvement in
the therapeutic effectiveness of SSRI in the patients with FM
Conclusion
This is to our knowledge the first observation that, apart from
a decrease in expression, there is also an alteration in the rate
of SERT which seems to depend not only on the SERT
number In fact, Bmax is not correlated with Vmax (data not
shown)
In the patients with FM, the decrease in 5-HT levels, which had
already been observed, together with the impaired SERT
func-tionality, might contribute to the pathogenesis of the disease,
both in quantity and rate In fact, these two factors are
impor-tant because they are correlated with the level of disease
severity
Thus, the results of the present study are in agreement with the
hypothesis that a deficit in the 5-HT transporter site could be
of pathogenetic significance in FM syndrome
Competing interests
The authors declare that they have no competing interests
Authors' contributions
L Bazzichi was responsible for the design of the study and
patient recruitment GG was responsible for the design of the
study and was the supervisor of the laboratory analysis LB
drafted the manuscript and was the coordinator of the
labora-tory activity GM performed the statistical analysis LF and PI
conducted the binding assays FDF and TG were responsible
for the uptake assays CG drafted the manuscript and
per-formed the statistical analysis AR was responsible for the
preparation of the samples and for their storage AL and SB
were general supervisors of the research group All authors
read and approved the final manuscript
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